The melanocortin circuitry of the CNS is a critical component of the adipostat. Activation of these circuits inhibits food intake and stimulates energy expenditure and thus the melanocortin-4 receptor has been a target of the major pharmaceutical companies for the development of drugs for the treatment of common obesity. Two clinical trials of potent MC4-R agonists exhibited unwanted pressor activity in some individuals. Recently, allosteric modulators of GPCRs have been recognized as a method of restoring normal spatio-temporal activity of physiological systems without the toxicity resulting from potent orthosteric agonists. Such allosteric modulators of the MC4R might have application to both severe syndromic obesity, as well as common obesity. Severe early onset obesity due to defective melanocortin signaling is linked, in up to 5% of cases, with non- synonymous coding mutations causing haploinsufficiency of the MC4R. It would not be unusual to expect that 10-30% of severe childhood obesity may thus result from defective melanocortin signaling, assuming MC4R promoter mutations, and mutations in other genes in the pathway may ultimately be discovered. The majority of MC4R mutations disrupt trafficking of receptors to the cell surface, rather than affinity for ligand. In contrast to common obesity, where excessive MC4R stimulation may cause unwanted side effects, successful treatment of severe obesity due to melanocortin receptor haploinsufficiency may involve increasing MC4R protein levels to physiological levels, thus potentially avoiding side effcets. Indeed, relative hypotension has been demonstrated in MC4R haploinsufficient obese patients. In this application, we propose to identify allosteric modulators of the MC4R, beginning with a high throughput screen of 162,000 compounds with the Vanderbilt High Throughput Screening Core. Initial hits will be validated, and a subset will be extensively characterized in cell culture models. Compound optimization will then be performed with the Vanderbilt Synthetic Chemistry Core, to develop preclinical lead compounds. We also propose to utilize two assays we present here, an electrophysiological slice preparation for MC4R function and a mouse model of MC4R haploinsufficiency, and to characterize antibodies against the MC4R to fully characterize the mechanism of action of allosteric modulators identified in the screen. Finally, we will attempt to identify serum biomarkers for melanocortin signaling as a less invasive tool for analysis of allosteric modulators of melanocortin signaling. PUBLIC HEALTH RELEVANCE: The melanocortin-4 receptor has been a target of the pharmaceutical industry for the development for therapeutics for the treatment of obesity because the receptor regulates body weight homeostasis, and haploinsufficiency of the receptor is the most common cause of severe early onset obesity. Clinical trials of potent orthosteric MC4-R agonists have not been successful, however, and pressor activity has been reported in at least some individuals in one trial. In this application we will conduct a high throughput screen with the Vanderbilt High Throughput Screening Facility, perform mechanism-of-action studies, and begin the process of optimization of hits with the Vanderbilt Synthetic Chemistry Core with the goal of identifying allosteric modulators of the MC4R that may maximize the normal activity of the receptor without tachyphylaxis or unwanted side effects.